Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu2+, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus

Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85-Å resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a β-sandwich of topology not previously described. A patch of conserved residues on the exposed face of a β-sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.

A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zα has allowed identification of motifs common to this class of nucleic acid binding domain.

Ku is associated with the telomere in mammals

Telomeres are specialized DNA/protein complexes that comprise the ends of eukaryotic chromosomes. The highly expressed Ku heterodimer, composed of 70 and 80 Kd subunits (Ku70 and Ku80), is the high-affinity DNA binding component of the DNA-dependent protein kinase. Ku is critical for nonhomologous DNA double-stranded break repair and site-specific recombination of V(D)J gene segments. Ku also plays an important role in telomere maintenance in yeast. Herein, we report, using an in vivo crosslinking method, that human and hamster telomeric DNAs specifically coimmunoprecipitate with human Ku80 after crosslinking. Localization of Ku to the telomere does not depend on the DNA-dependent protein kinase catalytic component. These findings suggest a direct link between Ku and the telomere in mammalian cells.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd ≈ 0.37 μM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI2) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI2 (Kd1 = 9.1 ± 2.0 nM; n1 = 170 ± 32 sites per cell vs. Kd1 = 7.2 ± 1.1 nM; n1 = 23 ± 8 sites per cell), with no significant change in the low-affinity receptors (Kd2 = 1.9 ± 0.1 μM; n2 = 1,832 ± 232 sites per cell vs. Kd2 = 1.6 ± 0.1 μM; n2 = 1,740 ± 161 sites per cell) as determined by Scatchard analysis of the binding of [3H]PGE1. The loss of high-affinity PGI2 binding led to the failure of PGI2 to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI2 to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [3H]PGE1 to the high-affinity PGI2 receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [3H]PGE1 to the high-affinity platelet PGI2 receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI2 binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.

DNA bending and the initiation of transcription at σ54-dependent bacterial promoters

We have examined the effects on transcription initiation of promoter and enhancer strength and of the curvature of the DNA separating these entities on wild-type and mutated enhancer–promoter regions at the Escherichia coli σ54-dependent promoters glnAp2 and glnHp2 on supercoiled and linear DNA. Our results, together with previously reported observations by other investigators, show that the initiation of transcription on linear DNA requires a single intrinsic or induced bend in the DNA, as well as a promoter with high affinity for σ54-RNA polymerase, but on supercoiled DNA requires either such a bend or a high affinity promoter but not both. The examination of the DNA sequence of all nif gene activator- or nitrogen regulator I-σ54 promoters reveals that those lacking a binding site for the integration host factor have an intrinsic single bend in the DNA separating enhancer from promoter.

A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxyl-terminus by ≈8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated (324 amino acids) rat SP receptors transfected stably into Chinese hamster ovary cells. Both the full-length and the truncated receptor bound radiolabeled SP with a similar Kd (≈0.1 nM). The average number of high affinity SP binding sites per cell was 1.0 × 105 and 0.3 × 105 for the full-length and the truncated SP receptor, respectively. In both cell lines, SP induced a rapid but transient increase in cytosolic calcium concentration ([Ca2+]i), which consisted of the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Both components are dependent on phospholipase C activation. Although the full-length and the truncated receptor utilize the same calcium pathways, they differ in their EC50 values (0.28 nM for the full-length; 0.07 nM for the truncated). These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.

Fe-catalyzed cleavage of the α subunit of Na/K-ATPase: Evidence for conformation-sensitive interactions between cytoplasmic domains

Incubation of Na/K-ATPase with ascorbate plus H2O2 produces specific cleavage of the α subunit. Five fragments with intact C termini and complementary fragments with intact N termini were observed. The β subunit is not cleaved. Cleavages depend on the presence of contaminant or added Fe2+ ions, as inferred by suppression of cleavages with nonspecific metal complexants (histidine, EDTA, phenanthroline) or the Fe3+-specific complexant desferrioxamine, or acceleration of cleavages by addition of low concentrations of Fe2+ but not of other heavy metal ions. Na/K-ATPase is inactivated in addition to cleavage, and both effects are insensitive to OH⋅ radical scavengers. Cleavages are sensitive to conformation. In low ionic strength media (E2) or media containing Rb ions [E2(Rb)], cleavage is much faster than in high ionic strength media (E1) or media containing Na ions (E1Na). N-terminal fragments and two C-terminal fragments (N-terminals E214 and V712) have been identified by amino acid sequencing. Approximate positions of other cleavages were determined with specific antibodies. The results suggest that Fe2+ (or Fe3+) ions bind with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe2+ (or Fe3+) ion. Thus, cleavage patterns can provide information on spatial organization of the polypeptide chain. We propose that highly conserved regions of the α subunit, within the minor and major cytoplasmic loops, interact in the E2 or E2(Rb) conformations but move apart in the E1 or E1Na conformations. We discuss implications of domain interactions for the energy transduction mechanism. Fe-catalyzed cleavages may be applicable to other P-type pumps or membrane proteins.

Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [3H]-5-HT-moduline bound specifically to rat brain sections with high affinity (Kd = 0.77 nM and Bmax = 0.26 dpm/mm2). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT1B receptor knock-out mice, the specific binding of [3H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.

hSK4, a member of a novel subfamily of calcium-activated potassium channels

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

Peptide analogue studies of the hypothalamic neuropeptide Y receptor mediating pituitary adrenocorticotrophic hormone release

Hypothalamic neuropeptide Y (NPY) is thought to be important in the regulation of feeding and also in the release of Adrenocorticotrophic hormone (ACTH). Intracerebroventricular administration of NPY to male rats significantly increased plasma ACTH 10 min after injection and stimulated 2-h food intake. A series of analogues of NPY that have a greatly reduced affinity for the Y1 [human pancreatic polypeptide (human PP), NPY(3–36)], the Y2 ([Pro34]NPY, human PP), the Y3 (peptide YY), and the Y6 (human PP) receptor, all markedly stimulated ACTH release. Rat PP, which binds with high affinity to the Y4 receptor, was unable to stimulate ACTH release. A novel analogue fragment [Pro34]NPY(13–36) was synthesized as a ligand with low Y1 and Y2 receptor affinity. Interestingly, neither [Pro34]NPY(13–36) nor the selective Y5 receptor agonist [d-Trp32]NPY stimulated food intake, whereas both significantly increased plasma ACTH. Thus the hypothalamic NPY receptor mediating increases in plasma ACTH has a fragment activation profile unlike the Y1–Y4 or Y6 receptors and appears distinct from the NPY receptor controlling food intake.

The amino-terminal region of Tyk2 sustains the level of interferon α receptor 1, a component of the interferon α/β receptor

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-α (IFN-α) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-α. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-α receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-α. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.